DNA is a complex macromolecule whose immunological activities are influenced by its base composition and base modification, as well as helical orientation. Certain unusual DNA structures (e.g., Z-DNA) can induce significant antibody responses when administered to normal mice. In addition, bacterial DNA, as well as certain synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine-guanine (CpG) sequences can induce cell proliferation and immunoglobulin (Ig) production in murine B cells.
Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than in vertebrates. Recent studies suggest that immune recognition of these motifs may contribute to the host's innate immune response. See, e.g., D. M. Klinman et al., CpG Motifs Present in Bacterial DNA Rapidly Induce Lymphocytes to Secrete Interleukin 6, Interleukin 12, and Interferon γ, 93 Proc. Natl. Acad. Sci. USA 2879 (1996); A.-K. Yi et al., Rapid Immune Activation by CpG Motifs in Bacterial DNA, 157 J. Immun. 5394 (1996); Hua Liang et al., Activation of Human B Cells by Phosphorothioate Oligodeoxynucleotides, 98 J. Clin. Invest. 1119 (1996); and A. M. Krieg et al., CpG Motifs in Bacterial DNA Trigger Direct B-Cell Activation, 374 Nature 546 (1995).
In mice, CpG oligonucleotides induces proliferation in almost all (>95%) B cells and increases Ig secretion. This B-cell activation by CpG DNA is T-cell independent and antigen non-specific. In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines. These cytokines stimulate natural killer (NK) cells to secrete γ-inteferon (IFN-γ) and have increased lytic activity. See, e.g., WO 95/26204, WO 96/02555, WO 98/11211, WO 98/18810, WO 98/37919, WO 98/40100, WO 98/52581 and U.S. Pat. No. 5,663,153.
The development of immunostimulatory CpG oligonucleotides and therapeutic and prophylactic methods of using them is ongoing. For example, U.S. Pat. No. 6,194,388 describes the administration of CpG oligonucleotides ex vivo, e.g., by obtaining lymphocytes from a subject and stimulating the subject's lymphocytes ex vivo upon contact with an appropriate oligonucleotide, and in vivo, e.g., by administering a non-methylated CpG containing oligonucleotide to a subject to facilitate in vivo activation of a subject's lymphocytes. According to U.S. Pat. No. 6,194,388, activated lymphocytes, stimulated by ex vivo or in vivo, can boost a subject's immune response. Hence, the immunostimulatory oligonucleotides can be used to treat, prevent or ameliorate an immune system deficiency (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection) in a subject. In addition, immunostimulatory oligonucleotides can also be administered as a vaccine adjuvant, to stimulate a subject's response to a vaccine.
U.S. Pat. No. 6,194,388 also suggests that immunostimulatory CpG oligonucleotides may be useful for treating leukemia by increasing the sensitivity of chronic leukemia cells toward conventional ablative chemotherapy. U.S. Pat. No. 6,194,388 also describes neutral oligonucleotides (i.e. oligonucleotide that do not contain an umethylated CpG or which contain a methylated CpG dinucleotide), which can compete for binding with unmethylated CpG containing oligonucleotides. According to U.S. Pat. No. 6,194,388, the in vivo administration of neutral oligonucleotides should prove useful for treating diseases such as systemic lupus erythematosus, sepsis and autoimmune diseases, which are caused or exacerbated by the presence of umethylated CpG dimers in a subject. Immunostimulatory CpG oligonucleotides and therapeutic uses thereof also are described in U.S. Patent Application Publication Nos. US 2003/0060440; US 2003/0144229; US 2004/0105872; US 2004/0241841; US 2004/02488334 and U.S. Pat. Nos. 6,207,646; 6,239,116; 6,339,068; 6,406,705; 6,653,292 and 6,727,230.
Positive results of human clinical and preclinical trials have been reported. Coley Pharmaceutical Group reported positive results in human clinical trials investigating the effectiveness of an immunostimulatory CpG oligonucleotide (CpG 7909) against various cancers, and also reported positive preclinical results of the effectiveness of CpG 7909 against bioterror agents such as anthrax in rodents and monkeys.
However, the cellular delivery of CpG oligonucleotides is generally inefficient, particularly for highly negatively charged CpG ODNs. Further, certain types of CpG oligonucleotides have a strong tendency to form tetrads, making large-scale commercial production impractical. Moreover, CpG oligonucleotides can be highly susceptible to nuclease cleavage, rendering them inactive, and CpG oligonucleotides have a limited duration of action.
In view of the foregoing, there exists a need for CpG oligonucleotides with improved cellular delivery and nuclease resistance profile. In addition there is a need for CpG oligonucleotides for which the duration and/or onset of action can be controlled. There is also a need for therapeutically effective CpG oligonucleotides, which avoids tetrad formation, e.g., during manufacture and/or storage. The present invention provides such CpG oligonucleotides, formulations thereof and therapeutic methods of using them. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.